uHTS fluorescence for the identification of compounds that decrease EGFP protein stability

Genetic studies have demonstrated that loss of the transcriptional co-activator p/CIP leads to resistance to obesity and diabetes, especially in extreme mouse obesity models. The goal of this research is to find small molecules that decrease p/CIP protein stability, thus providing new understanding and novel approaches to cure obesity and diabetes. In an in vitro white fat differentiation system, more ..

Genetic studies have demonstrated that loss of the transcriptional co-activator p/CIP leads to resistance to obesity and diabetes, especially in extreme mouse obesity models. The goal of this research is to find small molecules that decrease p/CIP protein stability, thus providing new understanding and novel approaches to cure obesity and diabetes. In an in vitro white fat differentiation system, p/CIP is important for fat cell formation. A fluorescence-based primary assay employing this in vitro cellular model has been developed to screen for small molecules that destabilize a p/CIP-EGFP fusion protein(AID 1984). As a counter screen to eliminate all the false positive hits that target only EGFP protein without affecting p/CIP, the same preadipocyte cell line was transfected with the same vector expressing EGFP only. The compounds that decrease levels of both EGFP and p/CIP-EGFP in counter screen and primary screen will be considered false positive, therefore eliminated from further testing.The assay described below is a cell-based HTS assay that utilizes anNIH3T3 derived subline F442A, a fibroblast line capable of differentiation into adipocytes in vitro transfected with an EGFP protein driven by a CMV promoter. The assay measures total fluorescence intensity of EGFP.

Compounds were tested in duplicate at 10 and 20 uM concentration. Compounds with a Z score of less than or equal to -3 OR >= 30% efficacy at 20 uM were defined as actives. ZScore is calculated as (wellValue - Mean) / StdDev. Where the mean is the mean of all of the valid wells on the plate and the STDDev is the standard deviation of all of the wells on the plateThe actives were subsequently run in dose response mode. Compounds with an IC50 < 40 uM were "actives" in the dose response screen.To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization is described below.1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 20 uM concentration:a. If primary % activity is less than 0%, then the assigned score is 0b. If primary % activity is greater than 100%, then the assigned score is 40c. If primary % activity is between 0% and 100%, then the calculated score is (% Inhibition)*0.42) Second tier (41-80 range) is reserved for dose-response confirmation data a. Inactive compounds of the confirmatory stage are assigned a score value equal 41. b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC: QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.d. Summary equation that takes into account the items discussed above is Score = 44 + 6*(pIC50 - 3)*QC, where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.3) Third tier (81-100 range) is reserved for re-synthesized true positives and their analogues and is not applicable in this assay